Journal of Cell Science

Src-mediated phosphorylation of mammalian Abp1 (DBNL) regulates podosome rosette formation in transformed fibroblasts.

Publication Date: 2012 Feb 2 PMID: 22303001
Authors: Boateng, L. R. - Cortesio, C. L. - Huttenlocher, A.
Journal: J Cell Sci

Podosomes are dynamic actin-based structures that mediate adhesion to the extracellular matrix and localize matrix degradation to facilitate cell motility and invasion. Drebrin-like protein (DBNL), which is homologous to yeast mAbp1 and is therefore known as mammalian actin-binding protein 1 (mAbp1), has been implicated in receptor-mediated endocytosis, vesicle recycling and dorsal ruffle formation. However, it is not known whether mAbp1 regulates podosome formation or cell invasion. In this study, we found that mAbp1 localizes to podosomes and is necessary for the formation of podosome rosettes in Src-transformed fibroblasts. Despite their structural similarity, mAbp1 and cortactin play distinct roles in podosome regulation. Cortactin was necessary for the formation of podosome dots, whereas mAbp1 was necessary for the formation of organized podosome rosettes in Src-transformed cells. We identified specific Src phosphorylation sites, Tyr337 and Tyr347 of mAbp1, which mediate the formation of podosome rosettes and degradation of the ECM. In contrast to dorsal ruffles, the interaction of mAbp1 with WASP-interacting protein (WIP) was not necessary for the formation of podosome rosettes. Finally, we showed that depletion of mAbp1 increased invasive cell migration, suggesting that mAbp1 differentially regulates matrix degradation and cell invasion. Collectively, our findings identify a role for mAbp1 in podosome rosette formation and cell invasion downstream of Src.

post to: CiteULike

Assembly of Bazooka polarity landmarks through a multifaceted membrane-association mechanism.

Publication Date: 2012 Feb 2 PMID: 22303000
Authors: McKinley, R. F. - Yu, C. G. - Harris, T. J.
Journal: J Cell Sci

Epithelial cell polarity is essential for animal development. The scaffold protein Bazooka (Baz/PAR-3) forms apical polarity landmarks to organize epithelial cells. However, it is unclear how Baz is recruited to the plasma membrane and how this is coupled with downstream effects. Baz contains an oligomerization domain, three PDZ domains, and binding regions for the protein kinase aPKC and phosphoinositide lipids. With a structure-function approach, we dissected the roles of these domains in the localization and function of Baz in the Drosophila embryonic ectoderm. We found that a multifaceted membrane association mechanism localizes Baz to the apical circumference. Although none of the Baz protein domains are essential for cortical localization, we determined that each contributes to cortical anchorage in a specific manner. We propose that the redundancies involved might provide plasticity and robustness to Baz polarity landmarks. We also identified specific downstream effects, including the promotion of epithelial structure, a positive-feedback loop that recruits aPKC, PAR-6 and Crumbs, and a negative-feedback loop that regulates Baz.

post to: CiteULike

Dynamics within tetraspanin pairs affect MHC class II expression.

Publication Date: 2012 Feb 2 PMID: 22302999
Authors: van den Hoorn, T. - Paul, P. - Janssen, L. - Janssen, H. - Neefjes, J.
Journal: J Cell Sci

Late endosomal multivesicular bodies (MVBs) are complicated organelles with various subdomains located at the limiting membrane and the internal vesicles (ILVs). ILVs accumulate tetraspanins such as CD63 and CD82 that might form protein assemblies, including major histocompatibility complex class II (MHC-II) and its chaperone human leukocyte antigen (HLA)-DM. Here, we studied the effect of four late endosomal tetraspanin proteins on MHC-II expression. Silencing CD9, CD63 and CD81 enhanced MHC-II expression whereas silencing CD82 did not. No effect on peptide loading was observed. Using confocal FRET technology, we measured the dynamics of CD63 and CD82 interaction with MHC-II and its chaperone HLA-DM. CD63-CD82 interactions remained unaltered in the two MVB subdomains whereas the interactions between CD63 or CD82 homologous pairs changed. CD63 stably associated with MHC-II, and CD82 with HLA-DM, on both MVB subdomains whereas the CD82-MHC-II and CD63-HLA-DM interactions changed. These data visualize for the first time the protein dynamics of tetraspanin assemblies in MVB subdomains. CD63, unlike CD82, stably interacts with MHC-II at both MVB subdomains and controls MHC-II expression.

post to: CiteULike

E-cadherin inhibits nuclear accumulation of Nrf2: implications for chemoresistance of cancer cells.

Publication Date: 2012 Feb 2 PMID: 22302998
Authors: Kim, W. D. - Kim, Y. W. - Cho, I. J. - Lee, C. H. - Kim, S. G.
Journal: J Cell Sci

Nrf2 has an anti-carcinogenic effect. However, an increase in Nrf2 activity is also implicated in cancer chemoresistance. A switch from E-cadherin to N-cadherin affects the transdifferentiation and metastasis of cancer cells. In view of the key role of this switch in cancer malignancy, we investigated the regulatory effect of E-cadherin on Nrf2. In HEK293 cells, overexpression of E-cadherin inhibited the nuclear accumulation of Nrf2, and prevented Nrf2-dependent gene induction. GST pull-down and immunocytochemical assays verified the interaction between E-cadherin and Nrf2: E-cadherin bound the C-terminus of Nrf2, but not its N-terminus, which comprises the Neh2 domain responsible for phosphorylation of Ser40. Our finding that the mutation of Ser40 to alanine in Nrf2 did not affect the ability of E-cadherin to bind Nrf2 and repress target gene transactivation suggests that E-cadherin might not disturb the phosphorylation. Studies using mutant constructs of E-cadherin suggested that the beta-catenin-binding domain contributes to the inhibitory effect of E-cadherin on Nrf2. Consistently, knockdown of beta-catenin attenuated not only the effect of E-cadherin binding to Nrf2, but also Keap1-dependent ubiquitylation of Nrf2, and thereby increased Nrf2 activity, supporting the involvement of beta-catenin in the interactions. Collectively, E-cadherin recruits Nrf2 through beta-catenin, and assists the function of Keap1 for the inhibition of nuclear localization and transcriptional activity of Nrf2. In HepG2 cells, the loss of E-cadherin by either siRNA knockdown or treatment with TGFbeta1 enhanced the constitutive or inducible activity of Nrf2, implying that chemoresistance of cancer cells upon the loss of E-cadherin might be associated with Nrf2.

post to: CiteULike

Flow cytometry analysis reveals a decrease in intracellular sodium during sperm capacitation.

Publication Date: 2012 Feb 2 PMID: 22302997
Authors: Escoffier, J. - Krapf, D. - Navarrete, F. - Darszon, A. - Visconti, P. E.
Journal: J Cell Sci

Mammalian sperm require time in the female tract in order to be able to fertilize an egg. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential. Recently, we presented evidence showing that epithelial Na(+) channels (ENaC) are present in mature sperm and that ENaCs are blocked during capacitation. In the present work, we used flow cytometry to analyze changes in intracellular Na(+) concentration ([Na(+)](i)) during capacitation in individual cells. Our results indicate that capacitated sperm have lower Na(+) concentrations. Using sperm with green fluorescent protein in their acrosomes, it was shown that the lower [Na(+)](i) concentration only occurs in sperm having intact acrosomes. ENaC inhibition has been shown in other cell types to depend on the activation of cystic fibrosis transmembrane conductance regulator (CFTR). In non-capacitated sperm, amiloride, an ENaC inhibitor, and genistein, a CFTR activator, caused a decrease in [Na(+)](i), suggesting that also in these cells [Na(+)](i) is dependent on the crosstalk between ENaC and CFTR. In addition, PKA inhibition blocked [Na(+)](i) decrease in capacitated sperm. Altogether, these data are consistent with the hypothesis that the capacitation-associated hyperpolarization involves a decrease in [Na(+)](i) mediated by inhibition of ENaC and regulated by PKA through activation of CFTR channels.

post to: CiteULike

Orai1 deficiency leads to heart failure and skeletal myopathy in zebrafish.

Publication Date: 2012 Feb 2 PMID: 22302996
Authors: Volkers, M. - Dolatabadi, N. - Gude, N. - Most, P. - Sussman, M. A. - Hassel, D.
Journal: J Cell Sci

Mutations in the store-operated Ca(2+) entry pore protein ORAI1 have been reported to cause myopathies in human patients but the mechanism involved is not known. Cardiomyocytes express ORAI1 but its role in heart function is also unknown. Using reverse genetics in zebrafish, we demonstrated that inactivation of the highly conserved zebrafish orthologue of ORAI1 resulted in severe heart failure, reduced ventricular systolic function, bradycardia and skeletal muscle weakness. Electron microscopy of Orai1-deficient myocytes revealed progressive skeletal muscle instability with loss of myofiber integrity and ultrastructural abnormalities of the z-disc in both skeletal and cardiac muscle. Isolated Orai1-deficient cardiomyocytes showed loss of the calcineurin-associated protein calsarcin from the z-discs. Furthermore, we found mechanosignal transduction was affected in Orai1-depleted hearts, indicating an essential role for ORAI1 in establishing the cardiac signaling transduction machinery at the z-disc. Our findings identify ORAI1 as an important regulator of cardiac and skeletal muscle function and provide evidence linking ORAI1-mediated calcium signaling to sarcomere integrity and cardiomyocyte function.

post to: CiteULike

GCP6 is a substrate of Plk4 and required for centriole duplication.

Publication Date: 2012 Feb 2 PMID: 22302995
Authors: Bahtz, R. - Seidler, J. - Arnold, M. - Haselmann-Weiss, U. - Antony, C. - Lehmann, W. D. - Hoffmann, I.
Journal: J Cell Sci

Centriole duplication occurs once per cell cycle and requires Plk4, a member of the Polo-like kinase family. A key component of the centrosome is the gamma-tubulin ring complex (gamma-TuRC) that nucleates microtubules. GCP6 is a member of the gamma-TuRC, but its role in human cells and the regulation of its functions remain unclear. Here we report that depletion of human GCP6 prevents assembly of the gamma-TuRC and induces a high percentage of monopolar spindles. These spindles are characterized by a loss of centrosomal gamma-tubulin and reduced centriole numbers. We found that GCP6 is localized in the pericentriolar material but also at distal portions of centrioles. In addition, GCP6 is required for centriole duplication and Plk4-induced centriole overduplication. GCP6 interacts with and is phosphorylated by Plk4. Moreover, we find that Plk4-dependent phosphorylation of GCP6 regulates centriole duplication. These data suggest that GCP6 is a target of Plk4 in centriole biogenesis.

post to: CiteULike

GBF1 (Gartenzwerg)-dependent secretion is required for Drosophila tubulogenesis.

Publication Date: 2012 Feb 2 PMID: 22302994
Authors: Wang, S. - Meyer, H. - Ochoa-Espinosa, A. - Buchwald, U. - Onel, S. - Altenhein, B. - J Heinisch, J. - Affolter, M. - Paululat, A.
Journal: J Cell Sci

Here we report on the generation and in vivo analysis of a series of loss-of-function mutants for the Drosophila ArfGEF, Gartenzwerg. The Drosophila gene gartenzwerg (garz) encodes the orthologue of mammalian GBF1. garz is expressed ubiquitously in embryos with substantially higher abundance in cells forming diverse tubular structures such as salivary glands, trachea, proventriculus or hindgut. In the absence of functional Garz protein, the integrity of the Golgi complex is impaired. As a result, both vesicle transport of cargo proteins and directed apical membrane delivery are severely disrupted. Dysfunction of the Arf1-COPI machinery caused by a loss of Garz leads to perturbations in establishing a polarized epithelial architecture of tubular organs. Furthermore, insufficient apical transport of proteins and other membrane components causes incomplete luminal diameter expansion and deficiencies in extracellular matrix assembly. The fact that homologues of Garz are present in every annotated metazoan genome indicates that secretion processes mediated by the GBF-type ArfGEFs play a universal role in animal development.

post to: CiteULike

NF-kappaB regulates protein quality control after heat stress through modulation of the BAG3-HspB8 complex.

Publication Date: 2012 Feb 2 PMID: 22302993
Authors: Nivon, M. - Abou-Samra, M. - Richet, E. - Guyot, B. - Arrigo, A. P. - Kretz-Remy, C.
Journal: J Cell Sci

We previously found that the NF-kappaB transcription factor is activated during the recovery period after heat shock; moreover, we demonstrated that NF-kappaB is essential for cell survival after heat shock by activating autophagy, a mechanism that probably helps the cell to cope with hyperthermic stress through clearance of damaged proteins. In this study, we analyze the involvement of NF-kappaB in basal and heat-stress-induced protein quality control, by comparing the level of multiubiquitylated and/or aggregated proteins, and proteasome and autophagic activity in NF-kappaB-competent and NF-kappaB-incompetent cells. We show that NF-kappaB has only a minor role in basal protein quality control, where it modulates autophagosome maturation. By contrast, NF-kappaB is shown to be a key player in protein quality control after hyperthermia. Indeed, NF-kappaB-incompetent cells show highly increased levels of multiubiquitylated and/or aggregated proteins and aggresome clearance defects; a phenotype that disappears when NF-kappaB activity is restored to normal. We demonstrate that during heat shock recovery NF-kappaB activates selective removal of misfolded or aggregated proteins - a process also called 'aggrephagy' - by controlling the expression of BAG3 and HSPB8 and by modulating the level of the BAG3-HspB8 complex. Thus NF-kappaB-mediated increase in the level of the BAG3-HspB8 complex leads to upregulation of aggrephagy and clearance of irreversibly damaged proteins and might increase cell survival in conditions of hyperthermia.

post to: CiteULike

Localization of glypican-4 in different membrane microdomains is involved in the regulation of Wnt signaling.

Publication Date: 2012 Feb 2 PMID: 22302992
Authors: Sakane, H. - Yamamoto, H. - Matsumoto, S. - Sato, A. - Kikuchi, A.
Journal: J Cell Sci

Glypicans are members of the heparan sulfate proteoglycans (HSPGs) and are involved in various growth factor signaling mechanisms. Although HSPGs affect the beta-catenin-dependent and -independent pathways of Wnt signaling, how they regulate distinct Wnt pathways is not clear. It has been suggested that the beta-catenin-dependent pathway is initiated through receptor endocytosis in lipid raft microdomains and the independent pathway is activated through receptor endocytosis in non-lipid raft microdomains. Here, evidence is presented that glypican-4 (GPC4) is localized to both membrane microdomains and that the localization affects its ability to regulate distinct Wnt pathways. GPC4 bound to Wnt3a and Wnt5a, which activate the beta-catenin-dependent and -independent pathways, respectively, and colocalized with Wnts on the cell surface. LRP6, one of Wnt3a coreceptors, was present in lipid raft microdomains, whereas Ror2, one of Wnt5a coreceptors, was localized to non-lipid raft microdomains. Expression of GPC4 enhanced the Wnt3a-dependent beta-catenin pathway and the Wnt5a-dependent beta-catenin-independent pathway, and knockdown of GPC4 suppressed both pathways. A GPC4 mutant that was localized to only non-lipid raft microdomains inhibited the beta-catenin-dependent pathway but enhanced the beta-catenin-independent pathway. These results suggest that GPC4 concentrates Wnt3a and Wnt5a to the vicinity of their specific receptors in different membrane microdomains, thereby regulating distinct Wnt signaling.

post to: CiteULike

A dual role for Rac1 GTPases in the regulation of cell motility.

Publication Date: 2012 Feb 2 PMID: 22302991
Authors: Filic, V. - Marinovic, M. - Faix, J. - Weber, I.
Journal: J Cell Sci

Rac proteins are the only canonical Rho family GTPases in Dictyostelium, where they act as key regulators of the actin cytoskeleton. To monitor the dynamics of activated Rac1 in Dictyostelium cells, a fluorescent probe was developed that specifically binds to the GTP-bound form of Rac1. The probe is based on the GTPase-binding domain (GBD) from PAK1 kinase, and was selected on the basis of yeast two-hybrid, GST pull-down and fluorescence resonance energy transfer assays. The PAK1 GBD localizes to leading edges of migrating cells and to endocytotic cups. Similarly to its role in vertebrates, activated Rac1 therefore appears to control de novo actin polymerization at protruding regions of the Dictyostelium cell. Additionally, we found that the IQGAP-related protein DGAP1, which sequesters active Rac1 into a quaternary complex with actin-binding proteins cortexillin I and cortexillin II, localizes to the trailing regions of migrating cells. Notably, PAK1 GBD and DGAP1, which both bind to Rac1-GTP, display mutually exclusive localizations in cell migration, phagocytosis and cytokinesis, and opposite dynamics of recruitment to the cell cortex upon stimulation with chemoattractants. Moreover, cortical localization of the PAK1 GBD depends on the integrity of the actin cytoskeleton, whereas cortical localization of DGAP1 does not. Taken together, these results imply that Rac1 GTPases play a dual role in regulation of cell motility and polarity in Dictyostelium.

post to: CiteULike

Direct role of Bardet-Biedl syndrome proteins in transcriptional regulation.

Publication Date: 2012 Feb 2 PMID: 22302990
Authors: Gascue, C. - Tan, P. L. - Cardenas-Rodriguez, M. - Libisch, G. - Fernandez-Calero, T. - Liu, Y. P. - Astrada, S. - Robello, C. - Naya, H. - Katsanis, N. - Badano, J. L.
Journal: J Cell Sci

Primary cilia are conserved organelles that play crucial roles as mechano- and chemosensors, as well as transducing signaling cascades. Consequently, ciliary dysfunction results in a broad range of phenotypes: the ciliopathies. Bardet-Biedl syndrome (BBS), a model ciliopathy, is caused by mutations in 16 known genes. However, the biochemical functions of the BBS proteins are not fully understood. Here we show that the BBS7 protein (localized in the centrosomes, basal bodies and cilia) probably has a nuclear role by virtue of the presence of a biologically confirmed nuclear export signal. Consistent with this observation, we show that BBS7 interacts physically with the polycomb group (PcG) member RNF2 and regulate its protein levels, probably through a proteasome-mediated mechanism. In addition, our data supports a similar role for other BBS proteins. Importantly, the interaction with this PcG member is biologically relevant because loss of BBS proteins leads to the aberrant expression of endogenous RNF2 targets in vivo, including several genes that are crucial for development and for cellular and tissue homeostasis. Our data indicate a hitherto unappreciated, direct role for the BBS proteins in transcriptional regulation and potentially expand the mechanistic spectrum that underpins the development of ciliary phenotypes in patients.

post to: CiteULike

Altered nanofeature size dictates stem cell differentiation.

Publication Date: 2012 Feb 2 PMID: 22302989
Authors: Zouani, O. F. - Chanseau, C. - Brouillaud, B. - Bareille, R. - Deliane, F. - Foulc, M. P. - Mehdi, A. - Durrieu, M. C.
Journal: J Cell Sci

The differentiation of stem cells can be modulated by physical factors such as the micro- and nano-topography of the extracellular matrix. One important goal in stem cell research is to understand the concept that directs differentiation into a specific cell lineage in the nanoscale environment. Here, we demonstrate that such paths exist by controlling only the micro- and nano-topography of polymer surfaces. Altering the depth (on a nanometric scale) of micro-patterned surface structures allowed increased adhesion of human mesenchymal stem cells (hMSCs) with specific differentiation into osteoblasts, in the absence of osteogenic medium. Small (10 nm) depth patterns promoted cell adhesion without noticeable differentiation, whereas larger depth patterns (100 nm) elicited a collective cell organization, which induced selective differentiation into osteoblast-like cells. This latter response was dictated by stress through focal-adhesion-induced reorganization of F-actin filaments. The results have significant implications for understanding the architectural effects of the in vivo microenvironment and also for the therapeutic use of stem cells.

post to: CiteULike

CFTR regulation in human airway epithelial cells requires integrity of the actin cytoskeleton and compartmentalized cAMP and PKA activity.

Publication Date: 2012 Feb 2 PMID: 22302988
Authors: Monterisi, S. - Favia, M. - Guerra, L. - Cardone, R. A. - Marzulli, D. - Reshkin, S. J. - Casavola, V. - Zaccolo, M.
Journal: J Cell Sci

The cystic fibrosis transmembrane conductance regulator (CFTR) mutation DeltaF508CFTR still causes regulatory defects when rescued to the apical membrane, suggesting that the intracellular milieu might affect its ability to respond to cAMP regulation. We recently reported that overexpression of the Na(+)/H(+) exchanger regulatory factor NHERF1 in the cystic fibrosis (CF) airway cell line CFBE41o- rescues the functional expression of DeltaF508CFTR by promoting F-actin organization and formation of the NHERF1-ezrin-actin complex. Here, using real-time FRET reporters of both PKA activity and cAMP levels, we find that lack of an organized subcortical cytoskeleton in CFBE41o- cells causes both defective accumulation of cAMP in the subcortical compartment and excessive cytosolic accumulation of cAMP. This results in reduced subcortical levels and increased cytosolic levels of PKA activity. NHERF1 overexpression in CFBE41o- cells restores chloride secretion, subcortical cAMP compartmentalization and local PKA activity, indicating that regulation of DeltaF508CFTR function requires not only stable expression of the mutant CFTR at the cell surface but also depends on both generation of local cAMP signals of adequate amplitude and activation of PKA in proximity of its target. Moreover, we found that the knockdown of wild-type CFTR in the non-CF 16HBE14o- cells results in both altered cytoskeletal organization and loss of cAMP compartmentalization, whereas stable overexpression of wt CFTR in CF cells restores cytoskeleton organization and re-establishes the compartmentalization of cAMP at the plasma membrane. This suggests that the presence of CFTR on the plasma membrane influences the cytoskeletal organizational state and, consequently, cAMP distribution. Our data show that a sufficiently high concentration of cAMP in the subcortical compartment is required to achieve PKA-mediated regulation of CFTR activity.

post to: CiteULike

Presenilin-2 regulates the degradation of RBP-Jk protein through p38 mitogen-activated protein kinase.

Publication Date: 2012 Feb 2 PMID: 22302987
Authors: Kim, S. M. - Kim, M. Y. - Ann, E. J. - Mo, J. S. - Yoon, J. H. - Park, H. S.
Journal: J Cell Sci

Transcriptional regulation performs a central role in Notch1 signaling by recombining binding protein Suppressor of Hairless (RBP-Jk) - a signaling pathway that is widely involved in determination of cell fate. Our earlier work demonstrated the possible regulation of the Notch1-RBP-Jk pathway through protein degradation of RBP-Jk; however, the potential regulator for the degradation of RBP-Jk remains to be determined. Here, we report that the expression of endogenous and exogenous RBP-Jk was increased significantly in cells treated with proteasome- and lysosome-specific inhibitors. The effects of these inhibitors on RBP-Jk occurred in a dose- and time-dependent manner. The level of RBP-Jk protein was higher in presenilin-2 (PS2)-knockout cells than in presenilin-1 (PS1)-knockout cells. Furthermore, the level of RBP-Jk was decreased by expression of PS2 in PS1 and PS2 double-knockout cells. We also found that PS1-knockout cells treated with a specific inhibitor of p38 mitogen-activated protein kinase delta (MAPK) had significantly increased levels of RBP-Jk. p38 MAPK phosphorylates RBP-Jk at Thr339 by physical binding, which subsequently induces the degradation and ubiquitylation of the RBP-Jk protein. Collectively, our results indicate that PS2 modulates the degradation of RBP-Jk through phosphorylation by p38 MAPK.

post to: CiteULike

Tiam1-regulated osteopontin in senescent fibroblasts contributes to the migration and invasion of associated epithelial cells.

Publication Date: 2012 Feb 2 PMID: 22302986
Authors: Liu, J. - Xu, K. - Chase, M. - Ji, Y. - Logan, J. K. - Buchsbaum, R. J.
Journal: J Cell Sci

The tumor microenvironment undergoes changes concurrent with neoplastic progression. Cancer incidence increases with aging and is associated with tissue accumulation of senescent cells. Senescent fibroblasts are thought to contribute to tumor development in aging tissues. We have shown that fibroblasts deficient in the Rac exchange factor Tiam1 promote invasion and metastasis of associated epithelial tumor cells. Here, we use a three-dimensional culture model of cellular invasiveness to outline several steps underlying this effect. We find that stress-induced senescence induces decreased fibroblast Tiam1 protein levels and increased osteopontin levels, and that senescent fibroblast lysates induce Tiam1 protein degradation in a calcium- and calpain-dependent fashion. Changes in fibroblast Tiam1 protein levels induce converse changes in osteopontin mRNA and protein. Senescent fibroblasts induce increased invasion and migration in co-cultured mammary epithelial cells. These effects in epithelial cells are ameliorated by either increasing fibroblast Tiam1 or decreasing fibroblast osteopontin. Finally, in seeded cell migration assays we find that either senescent or Tiam1-deficient fibroblasts induce increased epithelial cell migration that is dependent on fibroblast secretion of osteopontin. These findings indicate that one mechanism by which senescent fibroblasts promote neoplastic progression in associated tumors is through degradation of fibroblast Tiam1 protein and the consequent increase in secretion of osteopontin by fibroblasts.

post to: CiteULike

Neuropilin-2 regulates alpha6beta1 integrin in the formation of focal adhesions and signaling.

Publication Date: 2012 Feb 2 PMID: 22302985
Authors: Goel, H. L. - Pursell, B. - Standley, C. - Fogarty, K. - Mercurio, A. M.
Journal: J Cell Sci

The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells with the ECM. Using epithelial cells from breast tumors, we defined NRP2(high) and NRP2(low) populations that differed in integrin expression and adhesion to laminin. Specifically, the NRP2(high) population adhered more avidly to laminin and expressed high levels of the alpha6beta1 integrin than the NRP2(low) population. The NRP2(high) population formed numerous focal adhesions on laminin that were not seen in the NRP2(low) population. These results were substantiated using breast carcinoma cell lines that express NRP2 and alpha6beta1 integrin. Depletion experiments revealed that adhesive strength on laminin but not collagen is dependent on NRP2, and that VEGF is needed for adhesion on laminin. A specific interaction between NRP2 and alpha6beta1 integrin was detected by co-immunoprecipitation. NRP2 is necessary for focal adhesion formation on laminin and for the association of alpha6beta1 integrin with the cytoskeleton. NRP2 also facilitates alpha6beta1-integrin-mediated activation of FAK and Src. Unexpectedly, we discovered that NRP2 is located in focal adhesions on laminin. The mechanism by which NRP2 regulates the interaction of alpha6beta1 integrin with laminin to form focal adhesions involves PKC activation. Together, our data reveal a new VEGF-NRP2 signaling pathway that activates the alpha6beta1 integrin and enables it to form focal adhesions and signal. This pathway is important in the pathogenesis of breast cancer.

post to: CiteULike

Autophagy plays an essential role in the clearance of Pseudomonas aeruginosa by alveolar macrophages.

Publication Date: 2012 Feb 2 PMID: 22302984
Authors: Yuan, K. - Huang, C. - Fox, J. - Laturnus, D. - Carlson, E. - Zhang, B. - Yin, Q. - Gao, H. - Wu, M.
Journal: J Cell Sci

Intracellular bacteria have been shown to cause autophagy, which impacts infectious outcomes, whereas extracellular bacteria have not been reported to activate autophagy. Here, we demonstrate that Pseudomonas aeruginosa, a Gram-negative extracellular bacterium, activates autophagy with considerably increased LC3 punctation in both an alveolar macrophage cell line (MH-S) and primary alveolar macrophages. Using the LC3 Gly120 mutant, we successfully demonstrated a hallmark of autophagy, conjugation of LC3 to phosphatidylethanolamine (PE). The accumulation of typical autophagosomes with double membranes was identified morphologically by transmission electron microscopy (TEM). Furthermore, the increase of PE-conjugated LC3 was indeed induced by infection rather than inhibition of lysosome degradation. P. aeruginosa induced autophagy through the classical beclin-1-Atg7-Atg5 pathway as determined by specific siRNA analysis. Rapamycin and IFN-gamma (autophagy inducers) augmented bacterial clearance, whereas beclin-1 and Atg5 knockdown reduced intracellular bacteria. Thus, P. aeruginosa-induced autophagy represents a host protective mechanism, providing new insight into the pathogenesis of this infection.

post to: CiteULike

Schnurri regulates hemocyte function to promote tissue recovery after DNA damage.

Publication Date: 2012 Jan 24 PMID: 22275438
Authors: Kelsey, E. M. - Luo, X. - Buckner, K. - Jasper, H.
Journal: J Cell Sci

Tissue recovery after injury requires coordinated regulation of cell repair and apoptosis, removal of dead cells, and regeneration. A critical step in this process is the recruitment of blood cells that mediate local inflammatory and immune responses, promoting tissue recovery. Here we identify a new role for the transcriptional regulator Schnurri (Shn) in the recovery of UV-damaged Drosophila retina. Using an experimental paradigm that allows precise quantification of tissue recovery after a defined dose of UV, we find that Shn activity in the retina is required to limit tissue damage. This function of Shn relies on its transcriptional induction of the PDGF-like growth factor pvf-1, which signals to tissue-associated hemocytes. We show the pvf-1 receptor, PVR, acts in hemocytes to induce a macrophage-like morphology, and that this is required to limit tissue loss after irradiation. Our results identify a new Shn-regulated paracrine signaling interaction between damaged retinal cells and hemocytes ensures recovery and homeostasis of the challenged tissue.

post to: CiteULike

N-cadherin expression level modulates integrin-mediated polarity and strongly impacts on the speed and directionality of glial cell migration.

Publication Date: 2012 Jan 24 PMID: 22275437
Authors: Camand, E. - Peglion, F. - Osmani, N. - Sanson, M. - Etienne-Manneville, S.
Journal: J Cell Sci

Perturbation of cell polarity is a hallmark of cancer cells. In carcinomas, loss of epithelial E-cadherin contributes to the loss of cell polarity, and promotes Epithelial-Mesenchymal Transition and carcinoma infiltration. However, the contribution of classical cadherins to the development of non-epithelial tumours is less documented. We investigated the impact of N-cadherin expression level on the polarity and migration of normal and tumour glial cells. Low levels of N-cadherin were frequently observed in human glioma samples and purified glioma cells. Using a wound-healing assay, we show that decreased N-cadherin level promotes a faster and less directed migration both in normal and tumour cells. N-cadherin-mediated contacts control cell velocity and polarity through the regulation of focal adhesions. In cells expressing low levels of N-cadherin, small focal adhesions are present at the entire cell periphery of confluent cells and are not affected by wounding of the cell monolayer. In these conditions, wound-induced integrin-mediated recruitment of the small GTPase Cdc42, activation of the Cdc42-mediated polarity pathway and centrosome reorientation do not occur. Re-expression of N-cadherin in gliomas restores cell polarity and strongly reduces cell velocity, suggesting that loss of N-cadherin could contributes to the invasive capacity of tumour astrocytes.

post to: CiteULike

Melanoregulin regulates retrograde melanosome transport through interaction with the RILP{middle dot}p150Glued complex in melanocytes.

Publication Date: 2012 Jan 24 PMID: 22275436
Authors: Ohbayashi, N. - Maruta, Y. - Ishida, M. - Fukuda, M.
Journal: J Cell Sci

Melanoregulin (Mreg), a dilute suppressor gene product, has been implicated in the regulation of melanosome transport in mammalian epidermal melanocytes, because Mreg deficiency was found to restore peripheral melanosome distribution from perinuclear melanosome aggregation in Rab27A-deficient melanocytes. However, the function of Mreg in melanosome transport has remained unknown. Here we show that Mreg regulates microtubule-dependent retrograde melanosome transport through the dynein-dynactin motor complex. Mreg interacted with the C-terminal domain of RILP (Rab interacting lysosomal protein) and formed a complex with RILP and p150(Glued), a component of the dynein-dynactin motor complex, in cultured cells. Overexpression of Mreg, RILP, or both in normal melanocytes induced perinuclear melanosome aggregation, whereas knockdown of Mreg or functional disruption of the dynein-dynactin motor complex restored peripheral melanosome distribution in Rab27A-deficient melanocytes. These findings reveal a novel mechanism by which the dynein-dynactin motor complex recognizes Mreg on mature melanosomes through interaction with RILP and is involved in their centripetal movement.

post to: CiteULike

Structure-function relationship of the Polo-like kinase in Trypanosoma brucei.

Publication Date: 2012 Jan 24 PMID: 22275435
Authors: Yu, Z. - Liu, Y. - Li, Z.
Journal: J Cell Sci

Polo-like kinases (Plks) play multiple roles in mitosis and cytokinesis in eukaryotes and are characterized by the C-terminal Polo-box domain (PBD) implicated in binding to Plk substrates, targeting Plk, and regulating Plk activity. The Plk homolog in Trypanosoma brucei possesses a similar architecture, but it lacks the crucial residues involved in substrate binding and regulates cytokinesis but not mitosis. Despite these, little is known about the regulation of TbPLK and the role of the PBD in TbPLK localization and function. Here, we addressed the requirement of the kinase activity and the PBD for TbPLK localization and function through coupling RNAi of endogenous TbPLK with ectopic expression of TbPLK mutants. We demonstrate that the kinase activity and phosphorylation of two threonine residues, Thr198 and Thr202, in the activation loop (T-loop) of the kinase domain are essential for TbPLK function but not for TbPLK localization. Deletion of the PBD abolishes TbPLK localization, but the PBD itself is not correctly targeted, indicating that TbPLK localization requires both the PBD and the kinase domain. Surprisingly, the kinase domain of TbPLK, but not the PBD, binds to its substrates, TbCentrin2 and p110, suggesting that TbPLK may interact with its substrate through different mechanisms. Finally, the PBD interacts with the kinase domain of TbPLK and inhibits its activity, and this inhibition is relieved when Thr198 is phosphorylated. Together, these results suggest an essential role of T-loop phosphorylation in TbPLK activation and crucial roles of the PBD in regulating TbPLK activity and localization.

post to: CiteULike

Apico-basal elongation requires a Drebrin E/EB3 complex in columnar Human epithelial cells.

Publication Date: 2012 Jan 30 PMID: 22275434
Authors: Bazellieres, E. - Massey-Harroche, D. - Barthelemy-Requin, M. - Richard, F. - Arsanto, J. P. - Le Bivic, A.
Journal: J Cell Sci

Although columnar epithelial cells are known to acquire an elongated shape, the mechanisms involved in this morphological particularity have not yet been completely elucidated. Using the columnar human intestinal Caco2 cells, it was established here that the levels of Drebrin E, an actin-binding protein, increase in the terminal web both in vitro and in vivo during the formation of the apical domain. Drebrin E depletion was found to impair cell compaction and elongation processes in the monolayer without affecting the cell polarity or the formation of tight junctions. Decreasing the Drebrin E levels disrupted the normal sub-apical F-actin/Myosin IIB/ betaII Spectrin network and the apical accumulation of EB3, a microtubule plus-end binding protein. Decreasing the EB3 levels resulted in a similar elongation phenotype to that resulting from Drebrin E depletion, without affecting cell compaction processes or the pattern of F-actin/Myosin IIB distribution. In addition, EB3, Myosin IIB and betaII Spectrin were found to form a Drebrin E dependent complex. All in all, these data suggest this complex connects the F-actin and microtubule networks apically during epithelial cell morphogenesis, while Drebrin E also contributes to stabilizing the actin-based terminal web.

post to: CiteULike

A function for Rac1 in the terminal differentiation and pigmentation of hair.

Publication Date: 2012 Jan 30 PMID: 22275433
Authors: Behrendt, K. - Klatte, J. - Pofahl, R. - Bloch, W. - Smyth, N. - Tscharntke, M. - Krieg, T. - Paus, R. - Niessen, C. - Niemann, C. - Brakebusch, C. - Haase, I.
Journal: J Cell Sci

The small GTPase Rac1 is ubiquitiously expressed in proliferating and differentiating layers of the epidermis and hair follicles. Previously, Rac1 was shown to regulate stem cell behaviour in these compartments. We have asked whether Rac1 has, in addition, a specific, stem cell independent function in the regulation of terminal hair follicle differentiation. To address this we have expressed a constitutively active mutant of Rac1, L61Rac1, only in the basal epidermal layer and outer root sheath of mice with epidermis specific deletion of endogenous Rac1, which show severe hair loss. The resulting Rescue mice exhibited a hair coat throughout their lives. Therefore, expression of Rac1 activity in the keratin 14 positive compartment of the skin is sufficient for the formation of hair follicles and hair at normal quantity. The quality of hair formed in Rescue mice was, however, not normal. Rescue mice showed a grey, dull hair coat, whereas that of wild type and L61Rac1 transgenic mice was black and shiny. Hair analysis in Rescue mice revealed altered structures of the hair shaft and the cuticle and disturbed organization of medulla cells and pigment distribution. Disorganization of medulla cells correlates with the absence of cortical, keratin filled spikes that normally protrude from the cortex into the medulla. The desmosomal cadherin Dsc2, which normally decorates these protrusions, was found to be reduced or absent in the hair of Rescue mice. Our study demonstrates regulatory functions for Rac1 in hair structure formation and -pigmentation and thereby identifies, for the first time, a role for Rac1 in terminal differentiation.

post to: CiteULike

A Meiotic Actin Ring (MeiAR) Essential for Proper Sporulation in Fission Yeast.

Publication Date: 2012 Jan 24 PMID: 22275432
Authors: Yan, H. - Balasubramanian, M. K.
Journal: J Cell Sci

Sporulation is a unique form of cytokinesis that occurs following meiosis II in many yeasts, during which four daughter cells (spores) are generated within a single mother cell. Here we characterize the role of F-actin in the process of sporulation in the fission yeast Schizosaccharomyces pombe. As shown previously, we find that F-actin assembles into 4 ring structures per ascus, referred to as the MeiAR (meiotic actin ring). The actin nucleators Arp2/3 and formin-For3 assemble into ring structures that overlap with Meu14, a protein known to assemble into the so-called leading edge, a ring structure that is known to guide forespore membrane assembly. Interestingly, F-actin makes rings that occupy a larger region behind the leading edge ring. Time-lapse microscopy showed that the MeiAR assembles near the spindle pole bodies and undergoes an expansion in diameter during the early stages of meiosis II, followed by closure in later stages of meiosis II. MeiAR closure completes the process of forespore membrane assembly. Loss of MeiAR leads to excessive assembly of forespore membranes with a deformed appearance. The rate of closure of the MeiAR is dictated by the function of the Septation Initiation Network (SIN). We conclude that the MeiAR ensures proper targeting of the membrane biogenesis machinery to the leading edge, thereby ensuring the formation of spherically shaped spores.

post to: CiteULike

The role and mechanism of activin A in neurite outgrowth of embryonic dorsal root ganglia of chicken.

Publication Date: 2012 Jan 24 PMID: 22275431
Authors: Fang, L. - Wang, Y. N. - Cui, X. L. - Fang, S. Y. - Ge, J. Y. - Sun, Y. - Liu, Z. H.
Journal: J Cell Sci

Activin A, a member of transforming growth factor beta (TGF-beta) superfamily, plays essential role in neuron survival as a neurotrophic and neuroprotective factor in central nervous system. However, the effects and mechanisms of activin A on the neurite outgrowth of dorsal root ganglia (DRG) remain unclear. In the present study, we found that activin A is expressed in DRG collected from chicken embryos on day 8 (E8). Moreover, activin A induced neurite outgrowth of the primary cultured DRG and maintained long-time survival of monolayer-cultured DRG neurons during the observation for 10 days. Follistatin (FS), an activin-binding protein, significantly inhibited activin A-induced neurite outgrowth of DRG, but fails to influence the effect of nerve growth factor (NGF) on DRG neurite outgrowth. Furthermore, the results showed that activin A significantly up-regulated mRNA expressions of activin receptor type IIA (ActRIIA) and calcitonin gene-related peptide (CGRP) in DRG, and stimulated serotonin (5-HT) production from DRG, indicating that activin A may induce DRG neurite outgrowth via promoting CGRP expression and stimulating 5-HT release. These data suggest that activin A plays an important role in the development of DRG by autocrine/paracrine manner.

post to: CiteULike

The novel formin FMNL3 is a cytoskeletal regulator of angiogenesis.

Publication Date: 2012 Jan 24 PMID: 22275430
Authors: Hetheridge, C. - Scott, A. N. - Swain, R. K. - Copeland, J. W. - Higgs, H. N. - Bicknell, R. - Mellor, H.
Journal: J Cell Sci

The process of angiogenesis requires endothelial cells to undergo profound changes in shape and polarity. This must involve remodelling of the endothelial cell cytoskeleton; however, we know little of this process or of the proteins that control it. We used a co-culture assay of angiogenesis to examine the cytoskeleton of endothelial cells actively undergoing angiogenic morphogenesis. We find that elongation of endothelial cells during angiogenesis is accompanied by stabilisation of microtubules and their alignment into parallel arrays directed at the growing tip. In other systems, similar microtubule alignments are mediated by the formin family of cytoskeletal regulators. We screened a library of human formins and indentified the novel formin FMNL3/FRL2 as a critical regulator of endothelial cell elongation during angiogenesis. We show that activated FMNL3 triggers microtubule alignment and that FMNL3 is required for microtubule alignment during angiogenic morphogenesis. FMNL3 is highly-expressed in the endothelial cells of Zebrafish during development and embryos depleted for FMNL3 show profound defects in developmental angiogenesis that are rescued by expression of the human gene. We conclude that FMNL3 is a novel regulator of endothelial microtubules during angiogenesis and is required for the conversion of quiescent endothelial cells into their elongated angiogenic morphology.

post to: CiteULike

Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy.

Publication Date: 2012 Jan 24 PMID: 22275429
Authors: Itakura, E. - Kishi-Itakura, C. - Koyama-Honda, I. - Mizushima, N.
Journal: J Cell Sci

Mitochondria can be degraded by autophagy; this process is termed mitophagy. The Parkinson disease-associated ubiquitin ligase Parkin can trigger mitophagy of depolarized mitochondria. However, how the autophagy machinery is involved in this specific type of autophagy remains to be determined. It has been speculated that adaptor proteins such as p62 may mediate interaction between the autophagosomal LC3 family of proteins and ubiquitinated protein on mitochondria. Here, we describe our systematic analysis of the recruitment of Atg proteins in Parkin-dependent mitophagy. Structures containing upstream Atg proteins, including ULK1, Atg14, DFCP1, WIPI-1, and Atg16L1, can associate with depolarized mitochondria even in the absence of membrane-bound LC3. Atg9A structures are also recruited to these damaged mitochondria as well as the autophagosome formation site during starvation-induced canonical autophagy. At initial steps of Parkin-mediated mitophagy, the structures containing the ULK1 complex and Atg9A are independently recruited to depolarized mitochondria and both are required for further recruitment of downstream Atg proteins except LC3. Autophagosomal LC3 is important for efficient incorporation of damaged mitochondria into the autophagosome at a later stage. These findings suggest a process whereby the isolation membrane is generated de novo on damaged mitochondria as opposed to one where a preformed isolation membrane recognizes mitochondria.

post to: CiteULike

Regulation of local expression of cell adhesion and motility-related mRNAs in breast cancer cells by IMP1/ZBP1.

Publication Date: 2012 Jan 1 PMID: 22266909
Authors: Gu, W. - Katz, Z. - Wu, B. - Park, H. Y. - Li, D. - Lin, S. - Wells, A. L. - Singer, R. H.
Journal: J Cell Sci

Metastasis involves tumor cell detachment from the primary tumor, and acquisition of migratory and invasive capabilities. These capabilities are mediated by multiple events, including loss of cell-cell contact, an increase in focal adhesion turnover and failure to maintain a normal cell polarity. We have previously reported that silencing of the expression of the zipcode-binding protein IMP1/ZBP1 in breast tumor patients is associated with metastasis. IMP1/ZBP1 selectively binds to a group of mRNAs that encode important mediators for cell adhesion and motility. Here, we show that in both T47D and MDA231 human breast carcinoma cells IMP1/ZBP1 functions to suppress cell invasion. Binding of ZBP1 to the mRNAs encoding E-cadherin, beta-actin, alpha-actinin and the Arp2/3 complex facilitates localization of the mRNAs, which stabilizes cell-cell connections and focal adhesions. Our studies suggest a novel mechanism through which IMP1/ZBP1 simultaneously regulates the local expression of many cell-motility-related mRNAs to maintain cell adherence and polarity, decrease focal adhesion turnover and maintain a persistent and directional motility.

post to: CiteULike

Periostin modulates myofibroblast differentiation during full-thickness cutaneous wound repair.

Publication Date: 2012 Jan 1 PMID: 22266908
Authors: Elliott, C. G. - Wang, J. - Guo, X. - Xu, S. W. - Eastwood, M. - Guan, J. - Leask, A. - Conway, S. J. - Hamilton, D. W.
Journal: J Cell Sci

The matricellular protein periostin is expressed in the skin. Although periostin has been hypothesized to contribute to dermal homeostasis and repair, this has not been directly tested. To assess the contribution of periostin to dermal healing, 6 mm full-thickness excisional wounds were created in the skin of periostin-knockout and wild-type, sex-matched control mice. In wild-type mice, periostin was potently induced 5-7 days after wounding. In the absence of periostin, day 7 wounds showed a significant reduction in myofibroblasts, as visualized by expression of alpha-smooth muscle actin (alpha-SMA) within the granulation tissue. Delivery of recombinant human periostin by electrospun collagen scaffolds restored alpha-SMA expression. Isolated wild-type and knockout dermal fibroblasts did not differ in in vitro assays of adhesion or migration; however, in 3D culture, periostin-knockout fibroblasts showed a significantly reduced ability to contract a collagen matrix, and adopted a dendritic phenotype. Recombinant periostin restored the defects in cell morphology and matrix contraction displayed by periostin-deficient fibroblasts in a manner that was sensitive to a neutralizing anti-beta1-integrin and to the FAK and Src inhibitor PP2. We propose that periostin promotes wound contraction by facilitating myofibroblast differentiation and contraction.

post to: CiteULike

Chronic hypoxia compromises repair of DNA double-strand breaks to drive genetic instability.

Publication Date: 2012 Jan 1 PMID: 22266907
Authors: Kumareswaran, R. - Ludkovski, O. - Meng, A. - Sykes, J. - Pintilie, M. - Bristow, R. G.
Journal: J Cell Sci

Hypoxic cells have been linked to genetic instability and tumor progression. However, little is known about the exact relationship between DNA repair and genetic instability in hypoxic cells. We therefore tested whether the sensing and repair of DNA double-strand breaks (DNA-dsbs) is altered in irradiated cells kept under continual oxic, hypoxic or anoxic conditions. Synchronized G0-G1 human fibroblasts were irradiated (0-10 Gy) after initial gassing with 0% O(2) (anoxia), 0.2% O(2) (hypoxia) or 21% O(2) (oxia) for 16 hours. The response of phosphorylated histone H2AX (gamma-H2AX), phosphorylated ataxia telangiectasia mutated [ATM(Ser1981)], and the p53 binding protein 1 (53BP1) was quantified by intranuclear DNA repair foci and western blotting. At 24 hours following DNA damage, residual gamma-H2AX, ATM(Ser1981) and 53BP1 foci were observed in hypoxic cells. This increase in residual DNA-dsbs under hypoxic conditions was confirmed using neutral comet assays. Clonogenic survival was also reduced in chronically hypoxic cells, which is consistent with the observation of elevated G1-associated residual DNA-dsbs. We also observed an increase in the frequency of chromosomal aberrations in chronically hypoxic cells. We conclude that DNA repair under continued hypoxia leads to decreased repair of G1-associated DNA-dsbs, resulting in increased chromosomal instability. Our findings suggest that aberrant DNA-dsb repair under hypoxia is a potential factor in hypoxia-mediated genetic instability.

post to: CiteULike

Annexin A5 stimulates autophagy and inhibits endocytosis.

Publication Date: 2012 Jan 1 PMID: 22266906
Authors: Ghislat, G. - Aguado, C. - Knecht, E.
Journal: J Cell Sci

Macroautophagy is a major lysosomal catabolic process activated particularly under starvation in eukaryotic cells. A new organelle, the autophagosome, engulfs cytoplasmic substrates, which are degraded after fusion with endosomes and/or lysosomes. During a shotgun proteome analysis of purified lysosomal membranes from mouse fibroblasts, a Ca(2+)-dependent phospholipid-binding protein, annexin A5, was found to increase on lysosomal membranes under starvation. This suggests a role for this protein, an abundant annexin with a still unknown intracellular function, in starvation-induced lysosomal degradation. Transient overexpression and silencing experiments showed that annexin A5 increased lysosomal protein degradation, and colocalisation experiments, based on GFP sensitivity to lysosomal acidic pH, indicated that this was mainly the result of inducing autophagosome-lysosome fusion. Annexin A5 also inhibited the endocytosis of a fluid-phase marker and cholera toxin, but not receptor-mediated endocytosis. Therefore, we propose a double and opposite role of annexin A5 in regulating the endocytic and autophagic pathways and the fusion of autophagosomes with lysosomes and endosomes.

post to: CiteULike

The ESCRT machinery mediates polarization of fibroblasts through regulation of myosin light chain.

Publication Date: 2012 Jan 1 PMID: 22266905
Authors: Lobert, V. H. - Stenmark, H.
Journal: J Cell Sci

Recent evidence implicates the endosomal sorting complex required for transport (ESCRT) in the regulation of epithelial polarity in Drosophila melanogaster, but the mechanisms responsible for this action remain unclear. Here we show that ESCRTs determine cell orientation during directed migration in human fibroblasts. We find that endosomal retention of alpha5beta1 integrin and its downstream signaling effector Src in ESCRT-depleted cells is accompanied by the failure to activate myosin light chain kinase (MLCK), which thereby cannot phosphorylate myosin regulatory light chain (MRLC). Using this mechanism, ESCRT-depleted fibroblasts fail to orient their Golgi complex to undergo directional migration and show impaired focal adhesion turnover and increased spreading on fibronectin. Consistent with these findings, expression of a phosphomimetic mutant of MRLC in ESCRT-depleted cells restores normal phenotypes during cell spreading and orientation of the Golgi. These results suggest that, through their role in regulating integrin trafficking, ESCRTs regulate phosphorylation of MRLC and, subsequently, Golgi orientation and cell spreading.

post to: CiteULike

TOR complex 2 (TORC2) in Dictyostelium suppresses phagocytic nutrient capture independently of TORC1-mediated nutrient sensing.

Publication Date: 2012 Jan 1 PMID: 22266904
Authors: Rosel, D. - Khurana, T. - Majithia, A. - Huang, X. - Bhandari, R. - Kimmel, A. R.
Journal: J Cell Sci

The TOR protein kinase functions in two distinct complexes, TOR complex 1 (TORC1) and 2 (TORC2). TORC1 is required for growth in response to growth factors, nutrients and the cellular energy state; TORC2 regulates AKT signaling, which can modulate cytoskeletal polarization. In its ecological niche, Dictyostelium engulf bacteria and yeast for nutrient capture. Despite the essential role of TORC1 in control of cellular growth, we show that nutrient particle capture (phagocytosis) in Dictyostelium is independent of TORC1-mediated nutrient sensing and growth regulation. However, loss of Dictyostelium TORC2 components Rictor/Pia, SIN1/RIP3 and Lst8 promotes nutrient particle uptake; inactivation of TORC2 leads to increased efficiency and speed of phagocytosis. In contrast to phagocytosis, we show that macropinocytosis, an AKT-dependent process for cellular uptake of fluid phase nutrients, is not regulated by either of the TOR complexes. The integrated and balanced regulation of TORC1 and TORC2 might be crucial in Dictyostelium to coordinate growth and energy needs with other essential TOR-regulated processes.

post to: CiteULike

Sumoylated protein tyrosine phosphatase 1B localizes to the inner nuclear membrane and regulates the tyrosine phosphorylation of emerin.

Publication Date: 2012 Jan 20 PMID: 22266903
Authors: Yip, S. C. - Cotteret, S. - Chernoff, J.
Journal: J Cell Sci

Protein tyrosine phosphatase (PTP)1B is an abundant non-transmembrane enzyme that plays a major role in regulating insulin and leptin signaling. Recently, we reported that PTP1B is inhibited by sumoylation, and that sumoylated PTP1B accumulates in a perinuclear distribution, consistent with its known localization in the endoplasmic reticulum (ER) and the contiguous outer nuclear membrane. Here, we report that, in addition to its localization at the ER, PTP1B also is found at the inner nuclear membrane, where it is heavily sumoylated. We also find that PTP1B interacts with emerin, an inner nuclear membrane protein that is known to be tyrosine phosphorylated, and that PTP1B expression levels are inversely correlated with tyrosine phosphorylation levels of emerin. PTP1B sumoylation greatly increases as cells approach mitosis, corresponding to the stage where tyrosine phosphorylation of emerin is maximal. In addition, expression of a non-sumoylatable mutant of PTP1B greatly reduced levels of emerin tyrosine phosphorylation. These results suggest that PTP1B regulates the tyrosine phosphorylation of a key inner nuclear membrane protein in a sumoylation- and cell-cycle-dependent manner.

post to: CiteULike

Liprin-alpha controls stress fiber formation by binding to mDia and regulating its membrane localization.

Publication Date: 2012 Jan 1 PMID: 22266902
Authors: Sakamoto, S. - Ishizaki, T. - Okawa, K. - Watanabe, S. - Arakawa, T. - Watanabe, N. - Narumiya, S.
Journal: J Cell Sci

Regulation of the actin cytoskeleton is crucial for cell morphology and migration. mDia is an actin nucleator that produces unbranched actin filaments downstream of Rho. However, the mechanisms by which mDia activity is regulated in the cell remain unknown. We pulled down Liprin-alpha as an mDia-binding protein. The binding is mediated through the central region of Liprin-alpha and through the N-terminal Dia-inhibitory domain (DID) and dimerization domain (DD) of mDia. Liprin-alpha competes with Dia autoregulatory domain (DAD) for binding to DID, and binds preferably to the open form of mDia. Overexpression of a Liprin-alpha fragment containing the mDia-binding region decreases localization of mDia to the plasma membrane and attenuates the Rho-mDia-mediated formation of stress fibers in cultured cells. Conversely, depletion of Liprin-alpha by RNA interference (RNAi) increases the amount of mDia in the membrane fraction and enhances formation of actin stress fibers. Thus, Liprin-alpha negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region.

post to: CiteULike

Mutational analysis supports a core role for Drosophila alpha-Catenin in adherens junction function.

Publication Date: 2012 Jan 1 PMID: 22266901
Authors: Sarpal, R. - Pellikka, M. - Patel, R. R. - Hui, F. Y. - Godt, D. - Tepass, U.
Journal: J Cell Sci

alpha-catenin associates the cadherin-catenin complex with the actin cytoskeleton. alpha-catenin binds to beta-catenin, which links it to the cadherin cytoplasmic tail, and F-actin, but also to a multitude of actin-associated proteins. These interactions suggest a highly complex cadherin-actin interface. Moreover, mammalian alphaE-catenin has been implicated in a cadherin-independent cytoplasmic function in Arp2/3-dependent actin regulation, and in cell signaling. The function and regulation of individual molecular interactions of alpha-catenin, in particular during development, are not well understood. We have generated mutations in Drosophila alpha-Catenin (alpha-Cat) to investigate alpha-Catenin function in this model, and to establish a setup for testing alpha-Catenin-related constructs in alpha-Cat-null mutant cells in vivo. Our analysis of alpha-Cat mutants in embryogenesis, imaginal discs and oogenesis reveals defects consistent with a loss of cadherin function. Compromising components of the Arp2/3 complex or its regulator SCAR ameliorate the alpha-Cat loss-of-function phenotype in embryos but not in ovaries, suggesting negative regulatory interactions between alpha-Catenin and the Arp2/3 complex in some tissues. We also show that the alpha-Cat mutant phenotype can be rescued by the expression of a DE-cadherin::alpha-Catenin fusion protein, which argues against an essential cytosolic, cadherin-independent role of Drosophila alpha-Catenin.

post to: CiteULike

Keratinocyte-associated protein 2 is a bona fide subunit of the mammalian oligosaccharyltransferase.

Publication Date: 2012 Jan 1 PMID: 22266900
Authors: Roboti, P. - High, S.
Journal: J Cell Sci

The oligosaccharyltransferase (OST) complex catalyses the N-glycosylation of polypeptides entering the endoplasmic reticulum, a process essential for the productive folding and trafficking of many secretory and membrane proteins. In eukaryotes, the OST typically comprises a homologous catalytic STT3 subunit complexed with several additional components that are usually conserved, and that often function to modulate N-glycosylation efficiency. By these criteria, the status of keratinocyte-associated protein 2 (KCP2) was unclear: it was found to co-purify with the canine OST suggesting it is part of the complex but, unlike most other subunits, no potential homologues are apparent in Saccharomyces cerevisiae. In this study we have characterised human KCP2 and show that the predominant species results from an alternative initiation of translation to form an integral membrane protein with three transmembrane spans. KCP2 localises to the endoplasmic reticulum, consistent with a role in protein biosynthesis, and has a functional KKxx retrieval signal at its cytosolic C-terminus. Native gel analysis suggests that the majority of KCP2 assembles into a distinct ~500 kDa complex that also contains several bona fide OST subunits, most notably the catalytic STT3A isoform. Co-immunoprecipitation studies confirmed a robust and specific physical interaction between KCP2 and STT3A, and revealed weaker associations with both STT3B and OST48. Taken together, these data strongly support the proposal that KCP2 is a newly identified subunit of the N-glycosylation machinery present in a subset of eukaryotes.

post to: CiteULike

Loss of Scribble causes cell competition in mammalian cells.

Publication Date: 2012 Jan 1 PMID: 22250205
Authors: Norman, M. - Wisniewska, K. A. - Lawrenson, K. - Garcia-Miranda, P. - Tada, M. - Kajita, M. - Mano, H. - Ishikawa, S. - Ikegawa, M. - Shimada, T. - Fujita, Y.
Journal: J Cell Sci

In Drosophila, normal and transformed cells compete with each other for survival in a process called cell competition. However, it is not known whether comparable phenomena also occur in mammals. Scribble is a tumor suppressor protein in Drosophila and mammals. In this study we examine the interface between normal and Scribble-knockdown epithelial cells using Madin-Darby Canine Kidney (MDCK) cells expressing Scribble short hairpin RNA (shRNA) in a tetracycline-inducible manner. We observe that Scribble-knockdown cells undergo apoptosis and are apically extruded from the epithelium when surrounded by normal cells. Apoptosis does not occur when Scribble-knockdown cells are cultured alone, suggesting that the presence of surrounding normal cells induces the cell death. We also show that death of Scribble-knockdown cells occurs independently of apical extrusion. Finally, we demonstrate that apoptosis of Scribble-knockdown cells depends on activation of p38 mitogen-activated protein kinase (MAPK). This is the first demonstration that an oncogenic transformation within an epithelium induces cell competition in a mammalian cell culture system.

post to: CiteULike

Quantitative mapping of averaged focal adhesion dynamics in migrating cells by shape normalization.

Publication Date: 2012 Jan 1 PMID: 22250204
Authors: Mohl, C. - Kirchgessner, N. - Schafer, C. - Hoffmann, B. - Merkel, R.
Journal: J Cell Sci

The spatially ordered formation and disassembly of focal adhesions is a basic requirement for effective cell locomotion. Because focal adhesions couple the contractile actin-myosin network to the substrate, their distribution determines the pattern of traction forces propelling the cell in a certain direction. In the present study, we quantitatively analyzed the spatial patterning of cell-substrate adhesion in migrating cells by mapping averaged focal adhesion growth dynamics to a standardized cell coordinate system. These maps revealed distinct zones of focal adhesion assembly, disassembly and stability and were strongly interrelated with corresponding actin flow and traction force patterns. Moreover, the mapping technique enables precise detection of even minute responses of adhesion dynamics upon targeted signaling perturbations. For example, the partial inhibition of vinculin phosphorylation was followed by the reduced number of newly formed adhesions, whereas growth dynamics of existing adhesions remained unaffected.

post to: CiteULike

Lateral spacing of adhesion peptides influences human mesenchymal stem cell behaviour.

Publication Date: 2012 Jan 16 PMID: 22250203
Authors: Frith, J. E. - Mills, R. J. - Cooper-White, J. J.
Journal: J Cell Sci

Mesenchymal stem cells (MSCs) have attracted great interest in recent years for tissue engineering and regenerative medicine applications due to their ease of isolation and multipotent differentiation capacity. In the past, MSC research has focussed on the effects of soluble cues, such as growth factors and cytokines; however, there is now increasing interest in understanding how parameters such as substrate modulus, specific extracellular matrix (ECM) components and the ways in which these are presented to the cell can influence MSC properties. Here we use surfaces of self-assembled maleimide-functionalized polystyrene-block-poly(ethylene oxide) copolymers (PS-PEO-Ma) to investigate how the spatial arrangement of cell adhesion ligands affects MSC behaviour. By changing the ratio of PS-PEO-Ma in mixtures of block copolymer and polystyrene homopolymer, we can create surfaces with lateral spacing of the PEO-Ma domains ranging from 34 to 62 nm. Through subsequent binding of cysteine-GRGDS peptides to the maleimide-terminated end of the PEO chains in each of these domains, we are able to present tailored surfaces of controlled lateral spacing of RGD (arginine-glycine-aspartic acid) peptides to MSCs. We demonstrate that adhesion of MSCs to the RGD-functionalized block-copolymer surfaces is through specific attachment to the presented RGD motif and that this is mediated by alpha5, alphaV, beta1 and beta3 integrins. We show that as the lateral spacing of the peptides is increased, the ability of the MSCs to spread is diminished and that the morphology changes from well-spread cells with normal fibroblastic morphology and defined stress-fibres, to less-spread cells with numerous cell protrusions and few stress fibres. In addition, the ability of MSCs to form mature focal adhesions is reduced on substrates with increased lateral spacing. Finally, we investigate differentiation and use qRT-PCR determination of gene expression levels and a quantitative alkaline phosphatase assay to show that MSC osteogenesis is reduced on surfaces with increased lateral spacing while adipogenic differentiation is increased. We show here, for the first time, that the lateral spacing of adhesion peptides affects human MSC (hMSC) properties and might therefore be a useful parameter with which to modify hMSC behaviour in future tissue engineering strategies.

post to: CiteULike

Cdt1p, through its interaction with Mcm6p, is required for the formation, nuclear accumulation and chromatin loading of the MCM complex.

Publication Date: 2012 Jan 1 PMID: 22250202
Authors: Wu, R. - Wang, J. - Liang, C.
Journal: J Cell Sci

Regulation of DNA replication initiation is essential for the faithful inheritance of genetic information. Replication initiation is a multi-step process involving many factors including ORC, Cdt1p, Mcm2-7p and other proteins that bind to replication origins to form a pre-replicative complex (pre-RC). As a prerequisite for pre-RC assembly, Cdt1p and the Mcm2-7p heterohexameric complex accumulate in the nucleus in G1 phase in an interdependent manner in budding yeast. However, the nature of this interdependence is not clear, nor is it known whether Cdt1p is required for the assembly of the MCM complex. In this study, we provide the first evidence that Cdt1p, through its interaction with Mcm6p with the C-terminal regions of the two proteins, is crucial for the formation of the MCM complex in both the cytoplasm and nucleoplasm. We demonstrate that disruption of the interaction between Cdt1p and Mcm6p prevents the formation of the MCM complex, excludes Mcm2-7p from the nucleus, and inhibits pre-RC assembly and DNA replication. Our findings suggest a function for Cdt1p in promoting the assembly of the MCM complex and maintaining its integrity by interacting with Mcm6p.

post to: CiteULike

The MCM-associated protein MCM-BP is important for human nuclear morphology.

Publication Date: 2012 Jan 1 PMID: 22250201
Authors: Jagannathan, M. - Sakwe, A. M. - Nguyen, T. - Frappier, L.
Journal: J Cell Sci

Mini-chromosome maintenance complex-binding protein (MCM-BP) was discovered as a protein that is strongly associated with human MCM proteins, known to be crucial for DNA replication in providing DNA helicase activity. The Xenopus MCM-BP homologue appears to play a role in unloading MCM complexes from chromatin after DNA synthesis; however, the importance of MCM-BP and its functional contribution to human cells has been unclear. Here we show that depletion of MCM-BP by sustained expression of short hairpin RNA (shRNA) results in highly abnormal nuclear morphology and centrosome amplification. The abnormal nuclear morphology was not seen with depletion of other MCM proteins and was rescued with shRNA-resistant MCM-BP. MCM-BP depletion was also found to result in transient activation of the G2 checkpoint, slowed progression through G2 and increased replication protein A foci, indicative of replication stress. In addition, MCM-BP depletion led to increased cellular levels of MCM proteins throughout the cell cycle including soluble MCM pools. The results suggest that MCM-BP makes multiple contributions to human cells that are not limited to unloading of the MCM complex.

post to: CiteULike

A conserved membrane-binding domain targets proteins to organelle contact sites.

Publication Date: 2012 Jan 1 PMID: 22250200
Authors: Toulmay, A. - Prinz, W. A.
Journal: J Cell Sci

Membrane contact sites (MCSs), where the membranes of two organelles are closely apposed, are regions where small molecules such as lipids or calcium are exchanged between organelles. We have identified a conserved membrane-binding domain found exclusively in proteins at MCSs in Saccharomyces cerevisiae. The synaptotagmin-like-mitochondrial-lipid binding protein (SMP) domain is conserved across species. We show that all seven proteins that contain this domain in yeast localize to one of three MCSs. Human proteins with SMP domains also localize to MCSs when expressed in yeast. The SMP domain binds membranes and is necessary for protein targeting to MCSs. Proteins containing this domain could be involved in lipid metabolism. This is the first protein domain found exclusively in proteins at MCSs.

post to: CiteULike

The nucleoporin-like protein NLP1 (hCG1) promotes CRM1-dependent nuclear protein export.

Publication Date: 2012 Jan 1 PMID: 22250199
Authors: Waldmann, I. - Spillner, C. - Kehlenbach, R. H.
Journal: J Cell Sci

Translocation of transport complexes across the nuclear envelope is mediated by nucleoporins, proteins of the nuclear pore complex that contain phenylalanine-glycine (FG) repeats as a characteristic binding motif for transport receptors. CRM1 (exportin 1), the major export receptor, forms trimeric complexes with RanGTP and proteins containing nuclear export sequences (NESs). We analyzed the role of the nucleoporin-like protein 1, NLP1 (also known as hCG1 and NUPL2) in CRM1-dependent nuclear transport. NLP1, which contains many FG repeats, localizes to the nuclear envelope and could also be mobile within the nucleus. It promotes the formation of complexes containing CRM1 and RanGTP, with or without NES-containing cargo proteins, that can be dissociated by RanBP1 and/or the cytoplasmic nucleoporin Nup214. The FG repeats of NLP1 do not play a major role in CRM1 binding. Overexpression of NLP1 promotes CRM1-dependent export of certain cargos, whereas its depletion by small interfering RNAs leads to reduced export rates. Thus, NLP1 functions as an accessory factor in CRM1-dependent nuclear protein export.

post to: CiteULike

Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation.

Publication Date: 2012 Jan 1 PMID: 22250198
Authors: Del Puerto, A. - Diaz-Hernandez, J. I. - Tapia, M. - Gomez-Villafuertes, R. - Benitez, M. J. - Zhang, J. - Miras-Portugal, M. T. - Wandosell, F. - Diaz-Hernandez, M. - Garrido, J. J.
Journal: J Cell Sci

In adult brains, ionotropic or metabotropic purinergic receptors are widely expressed in neurons and glial cells. They play an essential role in inflammation and neurotransmission in response to purines secreted to the extracellular medium. Recent studies have demonstrated a role for purinergic receptors in proliferation and differentiation of neural stem cells although little is known about their role in regulating the initial neuronal development and axon elongation. The objective of our study was to investigate the role of some different types of purinergic receptors, P2Y1, P2Y13 and P2X7, which are activated by ADP or ATP. To study the role and crosstalk of P2Y1, P2Y13 and P2X7 purinergic receptors in axonal elongation, we treated neurons with specific agonists and antagonists, and we nucleofected neurons with expression or shRNA plasmids. ADP and P2Y1-GFP expression improved axonal elongation; conversely, P2Y13 and ATP-gated P2X7 receptors halted axonal elongation. Signaling through each of these receptor types was coordinated by adenylate cyclase 5. In neurons nucleofected with a cAMP FRET biosensor (ICUE3), addition of ADP or Blue Brilliant G, a P2X7 antagonist, increased cAMP levels in the distal region of the axon. Adenylate cyclase 5 inhibition or suppression impaired these cAMP increments. In conclusion, our results demonstrate a crosstalk between two metabotropic and one ionotropic purinergic receptor that regulates cAMP levels through adenylate cyclase 5 and modulates axonal elongation triggered by neurotropic factors and the PI3K-Akt-GSK3 pathway.

post to: CiteULike

The integrator complex is required for integrity of Cajal bodies.

Publication Date: 2012 Jan 1 PMID: 22250197
Authors: Takata, H. - Nishijima, H. - Maeshima, K. - Shibahara, K.
Journal: J Cell Sci

The nucleus in eukaryotic cells is a highly organized and dynamic structure containing numerous subnuclear bodies. The morphological appearance of nuclear bodies seems to be a reflection of ongoing functions, such as DNA replication, transcription, repair, RNA processing and RNA transport. The integrator complex mediates processing of small nuclear RNA (snRNA), so it might play a role in nuclear body formation. Here, we show that the integrator complex is essential for integrity of the Cajal body. Depletion of INTS4, an integrator complex subunit, abrogated 3'-end processing of snRNA. A defect in this activity caused a significant accumulation of the Cajal body marker protein coilin in nucleoli. Some fractions of coilin still formed nucleoplasmic foci; however, they were free of other Cajal body components, such as survival of motor neuron protein (SMN), Sm proteins and snRNAs. SMN and Sm proteins formed striking cytoplasmic granules. These findings demonstrate that the integrator complex is essential for snRNA maturation and Cajal body homeostasis.

post to: CiteULike

Soluble molecules are key in maintaining the immunomodulatory activity of murine mesenchymal stromal cells.

Publication Date: 2012 Jan 1 PMID: 22250196
Authors: Liu, H. - Lu, K. - Macary, P. A. - Wong, K. L. - Heng, A. - Cao, T. - Kemeny, D. M.
Journal: J Cell Sci

Mesenchymal stromal cells (MSCs) possess both immuno-privileged and immuno-inhibitory properties that contribute to their therapeutic effects. Ex vivo expansion is required to obtain sufficient cells for therapy, but might also alter their immunological properties. To date there has been no systematic study of MSC immunobiology during extended culture. Here, we demonstrate that both immuno-privilege and immunosuppressive properties of MSCs change with increasing passage. We demonstrate that although MSCs exhibit powerful immunosuppressive effects through secretion of transforming growth factor-beta (TGF-beta) and induction of interleukin-10, these effects are diminished by a concomitant increase in MSC immunogenicity. Interferon-gamma treatment for 3 days induced extendedly cultured MSCs to express significantly higher levels of major histocompatibility complex class I. In vivo, this results in cells that induce significant delayed-type hypersensitivity reactions in allogeneic recipients. Importantly, these effects are alleviated by isolation of the transplanted MSCs using a semi-permeable barrier. Under these conditions, even MSCs cultured through as many as 14 passages still exhibit immuno-inhibitory effects in vivo. Furthermore, the levels of anti-inflammatory molecule TGF-beta secreted by MSCs were maintained in the extended culture. These data shed light on the variable results of allogeneic MSCs in transplantation and suggest alternative strategies for prolonging the effect of allogeneic MSCs in cell-based therapy.

post to: CiteULike

Dyrk1A negatively regulates the actin cytoskeleton through threonine phosphorylation of N-WASP.

Publication Date: 2012 Jan 1 PMID: 22250195
Authors: Park, J. - Sung, J. Y. - Park, J. - Song, W. J. - Chang, S. - Chung, K. C.
Journal: J Cell Sci

Neural Wiskott-Aldrich syndrome protein (N-WASP) is involved in tight regulation of actin polymerization and dynamics. N-WASP activity is regulated by intramolecular interaction, binding to small GTPases and tyrosine phosphorylation. Here, we report on a novel regulatory mechanism; we demonstrate that N-WASP interacts with dual-specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A). In vitro kinase assays indicate that Dyrk1A directly phosphorylates the GTPase-binding domain (GBD) of N-WASP at three sites (Thr196, Thr202 and Thr259). Phosphorylation of the GBD by Dyrk1A promotes the intramolecular interaction of the GBD and verprolin, cofilin and acidic (VCA) domains of N-WASP, and subsequently inhibits Arp2/3-complex-mediated actin polymerization. Overexpression of either Dyrk1A or a phospho-mimetic N-WASP mutant inhibits filopodia formation in COS-7 cells. By contrast, the knockdown of Dyrk1A expression or overexpression of a phospho-deficient N-WASP mutant promotes filopodia formation. Furthermore, the overexpression of a phospho-mimetic N-WASP mutant significantly inhibits dendritic spine formation in primary hippocampal neurons. These findings suggest that Dyrk1A negatively regulates actin filament assembly by phosphorylating N-WASP, which ultimately promotes the intramolecular interaction of its GBD and VCA domains. These results provide insight on the mechanisms contributing to diverse actin-based cellular processes such as cell migration, endocytosis and neuronal differentiation.

post to: CiteULike

The p65 subunit of NF-kappaB and PARP1 assist Snail1 in activating fibronectin transcription.

Publication Date: 2011 Dec 15 PMID: 22223884
Authors: Stanisavljevic, J. - Porta-de-la-Riva, M. - Batlle, R. - de Herreros, A. G. - Baulida, J.
Journal: J Cell Sci

Snail1 is a transcriptional repressor of E-cadherin that triggers epithelial-mesenchymal transition (EMT). Here, we report assisted Snail1 interaction with the promoter of a typical mesenchymal gene, fibronectin (FN1), both in epithelial cells undergoing EMT and in fibroblasts. Together with Snail1, the p65 subunit of NF-kappaB and PARP1 bound to the FN1 promoter. We detected nuclear interaction of these proteins and demonstrated the requirement of all three for FN1 transcription. Moreover, other genes involved in cell movement mimic FN1 expression induced by Snail1 or TGF-beta1 treatment and recruit p65NF-kappaB and Snail1 to their promoters. The molecular cooperation between Snail1 and NF-kappaB in transcription activation provides a new insight into how Snail1 can modulate a variety of cell programs.

post to: CiteULike

The induction of a nucleoplasmic reticulum by prelamin A accumulation requires CTP:phosphocholine cytidylyltransferase-alpha.

Publication Date: 2011 Dec 15 PMID: 22223883
Authors: Goulbourne, C. N. - Malhas, A. N. - Vaux, D. J.
Journal: J Cell Sci

Farnesylated prelamin A accumulates when the final endoproteolytic maturation of the protein fails to occur and causes a dysmorphic nuclear phenotype; however, the morphology and mechanisms of biogenesis of these changes remain unclear. We show here that acute prelamin A accumulation after reduction in the activity of the ZMPSTE24 endoprotease by short interfering RNA knockdown, results in the generation of a complex nucleoplasmic reticulum that depends for its formation on the enzyme CTP:phosphocholine-cytidylyltransferase-alpha (CCT-alpha, also known as choline-phosphate cytidylyltransferase A). This structure can form during interphase, confirming that it is independent of mitosis and therefore not a consequence of disordered nuclear envelope assembly. Serial-section dual-axis electron tomography reveals that these invaginations can take two forms: one in which the inner nuclear membrane infolds alone with an inter membrane space interior, and the other in which an invagination of both nuclear membranes occurs, enclosing a cytoplasmic core. Both types of invagination can co-exist in one nucleus and both are frequently studded with nuclear pore complexes (NPC), which reduces NPC abundance on the nuclear surface.

post to: CiteULike

Regulation of an RNA granule during spermatogenesis: acetylation of MVH in the chromatoid body of germ cells.

Publication Date: 2011 Dec 15 PMID: 22223882
Authors: Nagamori, I. - Cruickshank, V. A. - Sassone-Corsi, P.
Journal: J Cell Sci

During mammalian spermatogenesis, the mouse VASA homolog (MVH; also known as DDX4), a germ-cell-specific DEAD-box type RNA-binding protein, localizes in a germline-specific RNA granule termed the chromatoid body (CB). Genetic analyses have revealed that MVH is essential for progression through spermatogenesis, although the molecular mechanisms of its function remain elusive. We found that the acetyltransferase Hat1, and its cofactor, p46, are specifically colocalized with MVH in the CB and acetylate MVH at Lys405, leading to inactivation of its RNA-binding activity. Notably, the acetylation is developmentally regulated, paralleling the temporally regulated colocalization of Hat1 and p46 in the CB. We have identified 858 mRNAs as MVH targets, a large proportion of which correspond to previously known translationally arrested genes. Importantly, eIF4B mRNA, a target of MVH, is selectively released from the MVH-ribonucleoprotein (RNP) complex when MVH is acetylated, paralleling an increase in eIF4B protein. These findings reveal a previously unknown signaling pathway that links acetylation to RNA processing in the control of spermatogenesis.

post to: CiteULike

MAL/MRTF-A controls migration of non-invasive cells by upregulation of cytoskeleton-associated proteins.

Publication Date: 2011 Dec 15 PMID: 22223881
Authors: Leitner, L. - Shaposhnikov, D. - Mengel, A. - Descot, A. - Julien, S. - Hoffmann, R. - Posern, G.
Journal: J Cell Sci

Monomeric actin regulates gene expression through serum response factor (SRF) by inhibiting its transcriptional coactivator myocardin-related transcription factor (MAL/MRTF). Many affected genes encode cytoskeletal components. We have analysed the migratory effects of actin-MAL signalling and of new target genes in non-invasive highly adherent cells. Expression of active MAL impaired migration of both fibroblasts and epithelial cells, whereas dominant-negative constructs and partial knockdown of MAL/MRTF enhanced motility. Knockdown of three newly characterised G-actin-regulated MAL targets, integrin alpha5, plakophilin 2 (Pkp2) and FHL1, enhanced cell migration. All three were upregulated by external stimulation through actin-MAL-SRF signalling, and MAL and SRF were inducibly recruited to cis-regulatory elements of the integrin alpha5 and Pkp2 genes. Finally, the reduced migration of epithelial cells stably expressing MAL was partially reversed by knockdown of Pkp2 and FHL1. We conclude that the actin-MAL pathway promotes adhesive gene expression, including integrin alpha5, Pkp2 and FHL1, and that this is anti-motile for non-invasive cells harbouring high basal activity.

post to: CiteULike

Positive charges on the translocating polypeptide chain arrest movement through the translocon.

Publication Date: 2011 Dec 15 PMID: 22223880
Authors: Fujita, H. - Yamagishi, M. - Kida, Y. - Sakaguchi, M.
Journal: J Cell Sci

Polypeptide chains synthesized by membrane-bound ribosomes are translocated through, and integrated into, the endoplasmic reticulum (ER) membrane by means of the protein translocation channel, the translocon. Positive charges on the nascent chain determine the orientation of the hydrophobic segment as it is inserted into the translocon and enhance the stop-translocation of translocating hydrophobic segments. Here we show that positive charges temporarily arrested ongoing polypeptide chain movement through the ER translocon by electrostatic interaction, even in the absence of a hydrophobic segment. The C-terminus of the polypeptide chain was elongated during the arrest, and then the full-length polypeptide chain moved through the translocon. The translocation-arrested polypeptide was not anchored to the membrane and the charges were on the cytoplasmic side of the membrane. The arrest effect was prevented by negatively charged residues inserted into the positive-charge cluster, and it was also suppressed by high salt conditions. We propose that positive charges are independent translocation regulators that are more active than previously believed.

post to: CiteULike

Cell metabolism affects selective vulnerability in PINK1-associated Parkinson's disease.

Publication Date: 2011 Dec 15 PMID: 22223879
Authors: Yao, Z. - Gandhi, S. - Burchell, V. S. - Plun-Favreau, H. - Wood, N. W. - Abramov, A. Y.
Journal: J Cell Sci

Mitochondrial dysfunction plays a primary role in the pathogenesis of Parkinson's disease (PD), particularly in autosomal recessive forms of the disease caused by mutations encoding PINK1. Although mitochondrial pathology can be demonstrated in many cell types, it is neurons that bear the brunt of cell death in PD. We studied the mitochondrial physiology of neurons and muscle cells with loss of function of the nuclear encoded mitochondrial protein PINK1. PINK1 is widely expressed in many types of tissues, but deficiency selectively induces death in neurons. We report here that the same genetic defect results in opposing phenotypes in different cell types, depending on the metabolic properties of the cell. Thus, PINK1-deficient myocytes exhibit high basal mitochondrial membrane potential (Deltapsim), whereas PINK1-deficient neurons have been shown to exhibit a low Deltapsim. PINK1 deficiency induces impaired respiration in both cell types, with a concomitant increase in glycolytic activity. We demonstrate that the high glycolytic capacity in myocytes compared with neurons enables them to produce more ATP and, therefore, compensates for the metabolic defects induced by PINK1 deficiency. Furthermore, the high Deltapsim generated in PINK1 knockout (KO) muscle mitochondria enables them to buffer cytosolic Ca(2+) fluxes, rendering them resistant to Ca(2+) stress effectively. Conversely, PINK1 KO neurons were previously shown to develop mitochondrial Ca(2+) overload and Ca(2+)-induced mitochondrial depolarisation. Prevention of Ca(2+) dysregulation in myocytes might therefore account for the sparing of these cells in PD.

post to: CiteULike